A tRNA-based multiplex sgRNA expression system in zebrafish and its application to generation of transgenic albino fish

Tomoya Shiraki,Koichi Kawakami

doi:10.1038/s41598-018-31476-5

Abstract

The CRISPR/Cas9 system can be introduced into zebrafish as transgenes. Namely, expression of single-guide RNA (sgRNA) and controlled expression of Cas9 in transgenic zebrafish enables the study of gene functions in specific cell types. This transgenic CRISPR/Cas9 approach would be more useful if multiple sgRNAs could be expressed simultaneously since we could knock-out a gene more efficiently or disrupt multiple genes simultaneously. Here we describe a novel system to express multiple sgRNAs efficiently in zebrafish, that relies on the endogenous tRNA processing machinery. We cloned nine endogenous zebrafish tRNA genes, fused them to sgRNAs, and demonstrated that an active sgRNA can be produced from a precursor transcript containing either of these tRNAs. To show a proof of principle, we constructed transgenic fish expressing Cas9 under the control of the ubiquitin promoter and a single transcript containing three distinct sgRNAs, that targeted the slc45a2 (albino) gene, fused to tRNAs under the control of the U6 promoter. We found that the Tg(ubb:SpCas9,u6c:3xslc45a2-sgRNA) harbored mutations in all of the target sites in the albino gene and showed nearly complete albino phenotypes, which were amenable to imaging experiments. Thus, the tRNA-based multiplex sgRNA expression system should facilitate gene knock-out studies in transgenic zebrafish.

Highlights

The clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas[9] system is a bacterial adaptive immune system and has been shown to function for genome engineering in many species[1,2,3,4,5]
We identified tRNA genes (1) whose tRNA species defined by anticodons and amino acid-based isotypes have a substantial number of genes and have the greatest gene number among their tRNA isotypes, (2) whose “Score” calculated by the tRNAscan-SE program[24] is the highest within the tRNA species, (3) that have neither the BsaI nor BseRI site, both used for singleguide RNA (sgRNA) cloning, and (4) that do not possess TTTT, which is the termination signal for RNA polymerase III (Pol III)
Our results demonstrated that the endogenous tRNA processing system could efficiently produce multiple functional sgRNAs from a single precursor transcript in zebrafish

Summary

Introduction

The clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas[9] system is a bacterial adaptive immune system and has been shown to function for genome engineering in many species[1,2,3,4,5]. The CRISPR/Cas[9] system greatly facilitates interrogation of genome functions owing to its simplicity, efficiency, and versatility. Co-delivery of Cas[9] and sgRNA into cells enables genome editing via a non-homologous end-joining (NHEJ) or homology-directed repair (HDR) mechanism. Injection of multiple sgRNAs enabled multiplex biallelic genome editing in the injected zebrafish[8,9]. It is desirable to efficiently express multiple sgRNAs that can target multiple sites in a gene or multiple genes in zebrafish

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